This is a competitive renewal application for an R01 grant. Our goal is to further increase our understanding of how HIV-1 uses coreceptors to enter cells. A specific emphasis will be on learning more about the mechanisms of resistance to small molecule CCR5 or CXCR4 inhibitors and the properties of resistant variants. Such studies will not only increase our knowledge of the HIV-1 replication cycle at the basic science level, they will also assist clinicians in their use of CCR5 inhibitors to treat HIV-1 infection. Building on our work in the general area of coreceptor biology during the past 14 years, we now propose the following research plan: In our first Specific Aim, we will investigate whether wild type and CCR5 inhibitor-resistant viruses interact with different sub-populations of CCR5 coreceptors. We will explore whether and how HIV-1 Env, monoclonal antibodies (MAbs) and small-molecule inhibitors interact with different CCR5 sub-populations. These sub-populations may arise through conformational interconversions, post-translational modifications, be located in distinct micro-domains on the cell surface, and have distinguishable links to G-proteins and the endocytic machinery. The sub-populations may be present in different proportions on diverse target cells. We will study how and where parental and inhibitor-resistant viruses interact with inhibitor-bound and -free CCR5 sub-populations. In Specific Aim 2, we propose to define how amino acid changes in the gp41 transmembrane protein of CCR5 inhibitor-resistant viruses affect the interaction of gp120 subunits with CCR5. We have described a resistant virus in which, unexpectedly, the critical changes were located in the gp41 fusion peptide, which is not known to interact with CCR5. We now have evidence for additional, resistance-associated changes in the N-terminal region of gp41. We will study the effects of these various changes on Env topology, CD4-Env interactions with CCR5, Env-mediated fusion rates, and the membrane location of the virus-cell fusion process. Our third Specific Aim is to assess whether Env-mediated signaling via CCR5 or CXCR4 is important for HIV-1 entry. We will use receptor-mutants defective for signal transduction functions, or constitutively active, and viruses that can enter cells in the presence of CCR5 or CXCR4 antagonists, to explore whether transmembrane signaling is essential for, or modifies, entry or post-entry events in the HIV-1 replication cycle.